dentfcaton, quantfcatoof phosphopeptdes and phosphoprotens The MS MS spectra had been searched wth the ProLucd algorthm created Yates lab 25 aganst aP rat database that was concatenated to a decoy database whch the sequence for each entry the orgnal database was reversed.The ntroductoof decoy database was employed to assess the false dscovery rate of spectra database matchng algorthm 26.To dentfy phosphopeptdes, the search parameters ncluded a dfferental modfcatooserne, threonne and tyrosne resdues of 79.9663 amu, ndcatng the addtoof phosphorous grouothose resdues.The database search results were assembled and ftered usng the DTASelect system 27.The assembled database matchng selleck chemical result fe was implemented to obtaquanttatve ratos betweethe 14and 15versoof every peptde usng the computer software Census28.Since the control and PCtreated neurons have been ndvdually mxed wth 15brahomogenate, the rato dfference betweecontrol and PCtreated sample for your similar phosphopeptde represented the dfference the phophorylatolevel on the dentfed ste.
For SAC experments, the peptde ratos betweethe lght andheavy sotopes had been drectly utilized as surrogates for proteexpressoratos betweethe handle and PCtreated neurons.MRM valdatoof phosphopeptde Flavopiridol ratos Teheavy argnne or lysne contanng phosphopeptdes had been syntheszed and mxed wth equal molar ratos, the mxture was utilized as spke nternal requirements.3 batches of neurons had been ether untreated or handled wth PCP, 100 ug of cells lysate have been spked wth 50 fmol in the teheavy specifications.Fve transtoons for each peptde had been selected for SRM, as well as the transtotables are lsted Table S5.A standard LC SRM experment were carried out wth a 90 mnute gradent delvered by a Donex nanohPLC pumwth a flow price of 300 nl mn.SRM were carried out oa Thermo TSQ Vantage Trple Quadrupole mass spectrometer.The nano electrospray onzatocondtons had been smar to MudPT experments, whe the TSQ condtons have been set as, Q1 peak wdth was 0.7, Q3 scawdth was 0.002 mass unt, as well as collsogass pressure was at one.2 mTor.
Statstcal and bonformatcs analyss Two batches from the experment had been carried out to make bologcal replcates.To assess the reproducbty of mass spectrometry measurement, two MudPT runs were performed the frst batch of samples.To test the sgnfcance of transformed phosphorylatoevents, ANOVA was used whethe phosphopeptdes had been quantfed the two bologcal replcates.Otherwse, Grubbs outler test was appled

to fnd sgnfcant outlers.the two cases the sgnfcance value utilised was 0.05.To confdently assgthe phosphorylatostes, a bnomal probabty based algorthm was applied 29.Only phosphorylatostes wth 0.05 have been further consdered for motf analyss.For these phosphorylatostes, Motf X algorthm 30 was appled to extract amno acd sequence patterns flankng the phosphorylatostes.